Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli.

Summary The tetracycline resistance (Tc) and raffi nose-hydrogen sulfide (Raf-Hys) characters of Escherichia coli D1021 are located on two compatible, conjugative fi^– plasmids named pRSDI and pRSD2, respectively. These were transferred together or separately to the isogenic background of E. coli K12 and isolated as transconjugants. Plasmid molecules isolated as covalently closed circular DNA have been analysed by buoyant density centrifugation, sucrose gradient sedimentation and electron microscopy. pRSDI (Tc) showed a buoyant density of 1.716 g/cm³ (56% GC), the open circular (OC) form had a sedimentation coefficient of 37s. If grown without tetracycline prior to isolation the contour length of most pRSDI molecules was 14.6 (±0.1) m (30.3 × 106 daltons) with a minor species of 13.9 (± 0.1) m owing to a small deletion. Pronounced length variation of pRSDI by growth in the presence of tetracycline owing to gene amplification is subject of a subsequent paper. In contrast, pRSD2 (Raf-Hys) molecules are stable under all conditions tested. The buoyant density was 1.713 g/cm³ (53% GC), the sedimentation coefficient of OGDNA was 58s and the contour length was 40.2 ± 0.6 m (83 × 106 daltons). During exponential growth one copy of pRSD2 per chromosome was found indicating stringent control of plasmid replication. At the onset of stationary growth the copy number rose from one to four per host chromosome indicating relaxation of the replication control. The level of plasmid-coded -galactosidase increases with copy numbers and has been used to test the gene dosage.